NGF) from seminal plasma origin enhances Corpus Luteum function in llamas regardless the preovulatory follicle diameter.

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Contents lists available at ScienceDirect

Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci

Ovulation-inducing factor (OIF/NGF) from seminal plasma origin enhances Corpus Luteum function in llamas regardless the preovulatory follicle diameter

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M. Silva a,b , C. Ulloa-Leal d,e , C. Norambuena a , A. Fernández d , G.P. Adams c , M.H. Ratto d,∗ a

Escuela de Medicina Veterinaria, Universidad Católica de Temuco, Temuco, Chile Núcleo de investigación en Producción Alimentaria, Universidad Católica de Temuco, Temuco, Chile c Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada d Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile e Programa de Doctorado en Ciencias Veterinarias, Escuela de Postgrado, Facultad de Ciencias Veterinarias, Universidad Austral de Chile, Valdivia, Chile

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Article history: Received 9 February 2014 Received in revised form 21 May 2014 Accepted 28 May 2014 Available online xxx

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Keywords: Llama Ovulation-inducing factor Nerve Growth Factor Corpus Luteum Follicles

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1. Introduction

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Ovulation-inducing factor (OIF) is a protein present in llama seminal plasma that has recently been identified as ␤-Nerve Growth Factor (NGF) and it induces not only a high rate of ovulation but also appears to have luteotrophic properties in this species. A 2-by-2 experimental design was used to determine the effect of treatments (OIF/NGF vs GnRH) and categories of preovulatory follicle diameter (7–10 vs >10 mm) on ovulation rate, CL diameter and function in llamas. Llamas (n = 32 llamas per group) were randomly assigned to receive an intramuscular dose of: (a) 1 mg purified OIF/NGF in the presence of a follicle of 7–10 mm in diameter; (b) 50 ␮g of GnRH in the presence of a follicle of 7–10 mm in diameter; (c) 1 mg purified OIF/NGF in the presence of a follicle >10 mm in diameter; (d) 50 ␮g of GnRH in the presence of a follicle >10 mm in diameter. Llamas were examined by ultrasonography every 12 h from treatment to Day 2 (Day 0 = treatment) to detect ovulation, and again on Day 8 to determine CL diameter. Ovulation rates did not differ among groups. There was an effect of preovulatory follicle size on Corpus Luteum diameter at Day 8 (P < 0.001), however plasma progesterone concentration (n = 15/per group) was higher (P < 0.05) in the OIF/NGF – than that of the GnRH – treated group by the same day. We conclude that OIF/NGF treatment enhances CL function regardless preovulatory follicle size at the time of treatment. © 2014 Published by Elsevier B.V.

Embryo mortality has been described as one of the main causes of poor reproductive efficiency in llamas and

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∗ Corresponding author. Tel.: +56 63 293063. E-mail address: [email protected] (M.H. Ratto).

alpacas. Embryonic losses as high as 50–80% have been reported by Day 30 and 80 of gestation respectively in alpacas (Bravo et al., 1987, 2010; Fernandez-Baca, 1970). We also have recently documented an embryo loss rate of 45.4% (49/108) in alpacas during the first 35 days after mating (Ratto et al., 2011b), which is similar to the 50% to 58% rate reported in earlier studies (Bravo and Sumar, 1985; Fernandez-Baca, 1970). Nutritional constraints, inadequate

http://dx.doi.org/10.1016/j.anireprosci.2014.05.012 0378-4320/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Silva, M., et al., Ovulation-inducing factor (OIF/NGF) from seminal plasma origin enhances Corpus Luteum function in llamas regardless the preovulatory follicle diameter. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.05.012

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mating systems, chromosomal aberrations and hormonal imbalance (i.e. progesterone deficiency) could be responsible for such embryo loss (Bravo et al., 2010; Sumar and 41 Adams, 2006). The Corpus Luteum (CL) is the main source 42 of progesterone in llamas and alpacas, allowing an ade43 quate establishment and maintenance of gestation (Sumar, 44 1983); therefore any factor that may affect CL function at 45 any stage of gestation will influence pregnancy rate and 46 overall fertility in this species. 47 The above evidence underlines the importance of study48 ing factors or conditions affecting early CL development 49 and function in llamas, since their understanding may con50 tribute to the improvement of reproductive efficiency in 51 this species. 52 Seminal plasma is generally considered as a transport 53 and survival medium to sperm; however, it is increas54 ingly recognized as a contributor to the reproductive 55 process in roles different from those previously mentioned 56 (Robertson, 2007). Seminal plasma contains diverse factors 57 that influence the environment of the female reproduc58 tive tract, including changes of the uterine immune system 59 and the induction of embryotrophic factors (Robertson and 60 Sharkey, 2001). For instance, seminal plasma influences the 61 sperm competence and survival, embryo development and 62 uterus receptivity in the mice (Robertson, 2007). The nat63 ural mating or intrauterine infusion of seminal plasma in 64 pigs shortens the interval from estrous to ovulation by 14 h 65 (Waberski et al., 1995, 2006; Weitze et al., 1990). Indeed, it 66 has been reported that seminal plasma has a luteotrophic 67 effect in pigs after intrauterine infusion (O Leary et al., 68 2001). 69 Studies conducted in llamas and alpacas (Adams et al., 70 2005; Ratto et al., 2005, 2006a,b) have unequivocally 71 described the presence of an ovulation-inducing factor 72 (OIF) in the seminal plasma of these species, and this has 73 been recently biochemical and functionally characterized 74 (Ratto et al., 2010, 2011a; Silva et al., 2011a,b; 2012). Based 75 on these studies OIF is described as a potent ovulatory 76 molecule which has a luteotrophic effect after intramuscu77 lar or intrauterine infusion in llamas and alpacas (Adams 78 et al., 2005; Berland et al., 2013; Silva et al., 2011a). 79 In a recent llama study (Ratto et al., 2012) the com80 plete molecular characterization and identification of OIF 81 was accomplished, describing it as a neurotrophin known 82 as Nerve Growth Factor (␤-NGF). For the purposes of this 83 study, OIF/NGF will be used to denote llama ␤-NGF from 84 seminal plasma origin. 85 The ovulation-inducing mechanism of OIF/NGF is 86 mediated by a direct effect at the central level of hypothala87 mic–pituitary axis, stimulating pituitary LH secretion by a 88 previous action on hypothalamic GnRH neurons (Silva et al., 89 2011a), in a dose response manner (Tanco et al., 2011), and 90 partially modulated by peripheral concentration of estra91 diol (Silva et al., 2012). 92 Several llama studies strongly suggest that this protein 93 may have luteotrophic properties (Adams et al., 2005; Silva 94 95Q3 et al., 2011a; Ulloa et al., 2013), since CLs developed after OIF/NGF administration consistently secrete more proges96 terone than those induced after GnRH treatment. However, 97 if this effect is dependent on preovulatory follicle (POF) size 98 has not been studied. 99 39 40

In this regard, a positive relationship between the diameter of the ovulatory follicle and subsequent CL size and plasma progesterone concentration has been reported in ruminants, such as cattle (Busch et al., 2008; Colazo et al., 2009; Perry et al., 2005), buffalo (Pandey et al., 2011) and sheep (Murdoch and Van Kirk, 1998). In llamas, a robust effect of follicle size on the pituitary and ovarian response to copulation has been described (Bravo et al., 1991), with a minimum POF diameter of 7 mm being necessary for ovulation to occur. Also, the characteristics of CL development after natural induction (i.e. mating) of ovulation is well described for this species (Aba et al., 1995; Adams et al., 1990, 1991; Ratto et al., 2006a). However, no studies have been designed to determine the relationship between the diameter of the preovulatory follicle and subsequent CL size and function in neither llamas nor alpacas. Therefore, a field study was conducted to determine the effect of the preovulatory follicle size or treatment (OIF/NGF vs GnRH) on ovulation rate and CL diameter and function. We hypothesize that preovulatory follicle diameter is positively associated to subsequent CL diameter and plasma progesterone concentration, and that CL development and function is enhanced by the administration of purified OIF/NGF from llama seminal plasma. 2. Materials and methods The study was conducted at the Universidad Austral de Chile, Valdivia, Chile (39◦ 38 S–73◦ 5 W and 19 m above sea level). All procedures were reviewed and approved by the Bioethics Committee and were performed in accordance with the animal care protocols established by the Universidad Austral de Chile. 2.1. Animals Non pregnant, non-lactating adult llamas (n = 128), ≥4 y of age and weighing 90–135 kg were maintained on pasture supplemented with hay and water ad libitum. Llamas were housed indoors at night and offered 200 g/animal of a commercial diet supplement containing 140 g/kg crude protein and 150 g/kg crude fiber. Additionally, adult male llamas (n = 4) ≥5 y of age and weighing 100–145 kg were maintained separately under similar conditions. 2.2. Seminal plasma collection and Protein purification Semen from 4 adult male llamas was collected twice per week for three months prior to the start of the study. Semen was collected with the use of an artificial vagina designed for sheep that was fitted into a phantom mount built of wood and covered with a llama hide (Bravo et al., 1997). An average of 20 ejaculates was collected from each male. Llama semen processing and purification of OIF/NGF has been described elsewhere (Silva et al., 2012; Tanco et al., 2011). In brief, semen was diluted with phosphate buffered saline (PBS, Gibco, Grand Island, NY, USA) and centrifuged for 30 min at 1500 × g at room temperature. The supernatant was decanted to remove sperm and spermfree seminal plasma was stored at −80 ◦ C. Upon thawing,


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the diluted seminal plasma was pooled, sonicated to reduce viscosity and centrifuged at 10,000 × g for 20 min to remove particulate matter. Purification of OIF/NGF was performed in a 2-step procedure as previously described (Silva et al., 2012; Tanco et al., 2011). Llama seminal plasma was loaded into a Type 1 macro-prep ceramic hydroxyapatite column (1 cm × 10 cm, 20 ␮m, BIO-RAD laboratories, Hercules, CA, USA) previously equilibrated with 10 mM sodium phosphate at a pH of 6.8. Elution was carried out at room temperature using a linear gradient with 350 mM sodium phosphate, pH 6.8, and a flow rate of 0.5 ml/min. An eluted fraction showing a major protein on SDS-PAGE, was concentrated in phosphate buffered saline (PBS, pH 7.4) using a 5 kDa cut-off membrane filter device (Vivaspin, Sartorius, Goettingen, Germany) and subsequently loaded onto a gel filtration column (SEC, Hi PrepTM 26/60 SepahacrylTM S-100, Amersham Laboratories, Piscataway, NJ, USA). The purification procedure was carried out at room temperature at a flow rate of 0.5 ml/min using fast protein liquid chromatography (FPLC, Amersham Laboratories, Piscataway, NJ, USA). Elution was performed isocratically using PBS at pH 7.4. The bioactive fraction after gel filtration was identified and defined as purified llama OIF/NGF.

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2.3. Experimental design

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Llamas were examined daily using transrectal ultrasonography with a 7.5 MHz linear-array transducer (AlokaSSD 500; Aloka Co., Tokyo, Japan) to monitor follicular growth. Females with the presence of a growing follicle ≥7 mm in diameter that had grown for 5 consecutive days were selected for the study (Adams et al., 2005; Ratto et al., 2005, 2006a,b). A 2-by-2 design was used involving treatment with OIF/NGF vs GnRH, and two categories of preovulatory follicle diameter (7–10 vs >10 mm). Female llamas were assigned randomly to four groups (n = 32 females per group) and given an intramuscular dose of: (a) 1 mg purified OIF/NGF with the presence of a growing follicle of 7–10 mm in diameter; (b) 1 mg purified OIF/NGF with the presence of a growing follicle greater than 10 mm in diameter; (c) 50 ␮g of GnRH (Ovalyse® , gonadoreline acetate, Pfizer Chile S.A., Santiago, Chile) with the presence of a growing follicle of 7–10 mm in diameter; or (d) 50 ␮g of GnRH with the presence of a growing follicle greater than 10 mm in diameter. Day of treatment was designated as Day 0.

2.4. Ultrasonographic examination Llamas were examined by ultrasonography every 12 h from treatment to Day 2 to detect ovulation, and again on Day 8 to determine CL diameter, based on previous reports consistently describing the greatest CL diameter by this day (Adams et al., 1991, 2005; Silva et al., 2011a). Ovulation was defined as the sudden disappearance of a large follicle ≥7 mm that was detected during the previous examination, and was confirmed by ultrasonographic detection of a CL 8 d after treatment.

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2.5. Blood collection and hormone assays In a subset of ovulated llamas (n = 15 llamas per group) blood samples for progesterone measurement were taken on Day 8, based on earlier studies describing the highest blood plasma progesterone concentration on this day (Aba et al., 1995, 2000; Adams et al., 1991). Blood samples were collected into heparinized tubes (Vacutainer Systems, Becton Dickinson, Franklin Lakes, NJ, USA) by jugular venipuncture. Blood was centrifuged for 10 min at 1500 × g and plasma was stored at −20 ◦ C. Plasma progesterone concentration was determined using a commercial, double-antibody radioimmunoassay kit (Coat-a-Count total progesterone, DPC; Diagnostic Products Corporation, Los Angeles, CA, USA) as previously reported (Adams et al., 2005; Silva et al., 2011a,b). The intra-assay coefficients of variation were 3.2, 2.9 and 3.6% for the low-, medium-, and high-reference plasma (means: 1.8, 3.5, and 16.5 ng/ml). The inter-assay coefficients of variation for low-, medium-, and high-reference plasma were 7.0, 9.0 and 6.0%, respectively. The sensitivity of the assay was 0.1 ng/ml. 2.6. Statistical analyses Statistical analyses were performed using the Statistical Analysis System software package (SAS Learning Edition, version 4.1, SAS Institute Inc., Cary, NC, USA, 2006). Ovulation rates were compared among groups by Chi-square, and CL diameter and plasma progesterone concentration at Day 8 were analyzed using a 2-by-2 factorial design. The analysis included the main effects of treatment (OIF/NGF vs GnRH), a preovulatory follicle category (7–10 vs >10 mm) and their interaction. If significant (P ≤ 0.05) main effects or interactions were detected, Bonferroni’ post hoc test for multiple comparisons was used to locate differences. If main effects were not detected data were grouped based on POF diameter category and treatment, and analyzed by a Student’s t-test. All data are reported as mean ± SEM, and probabilities ≤0.05 were considered significant, whereas P > 0.05 but ≤0.1 were considered trends approaching significance. 3. Results Although, the mean size of preovulatory follicle did differ among groups, there was not a significant difference in the ovulatory response among groups after treatment (P = 0.9; Table 1). Corpus Luteum (mean ± SEM) diameter at Day 8 was affected (P < 0.0001, Table 1) by the preovulatory follicle category, but there was no effect of treatment (P = 0.88) or their interaction (P = 0.4) on CL diameter by the same day. Since treatment did not affect CL size at Day 8 data from llamas treated with either OIF/NGF or GnRH were combined based on POF size (Fig. 1). Plasma progesterone concentration (mean ± SEM) was affected (P < 0.05) by treatment at Day 8, but no effect of preovulatory follicle category (P = 0.73) or their interaction (P = 0.5) was detected. Plasma progesterone concentration was higher in the OIF/NGF – than in the GnRH – treated group (Table 1); therefore, data from llamas with either


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Table 1 Effect of preovulatory follicle category (size) on ovarian and endocrine response in llamas treated with OIF/NGF or GnRH (Day 0 = day of treatment). End point/Treatment

Preovulatory follicle category 7–10 mm

POF diameter (mm) Ovulation rate (%) CL diameter at Day 8 (mm)* Progesterone concentration at Day 8 (ng/ml)** , a b c d * ** +

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GnRH (n = 32)

OIF/NGF (n = 32)

GnRH (n = 32)

8.8 ± 0.2a 27/30 (90) 12.5 ± 0.3c 6.3 ± 0.6a

9.1 ± 0.1a 32/34 (94.1) 12.3 ± 0.3c 4.6 ± 0.5b

12.0 ± 0.3b 32/34 (94.1) 13.5 ± 0.3d 5.7 ± 0.7a

11.8 ± 0.2b 25/28 (89.3) 13.8 ± 0.2d 4.9 ± 0.6b

Within rows values with no common superscript are different (P < 0.05). Within rows values with no common superscript are different (P < 0.05). Within rows values with no common superscript are different (P < 0.001). Within rows values with no common superscript are different (P < 0.001). Effect of preovulatory follicle category (P < 0.0001), effect of treatment (P = 0.88), interaction (P = 0.4). Effect of treatment (P < 0.05), effect of preovulatory follicle category (P = 0.73), interaction (P = 0.5). n = 15 llamas per group.

Fig. 1. Mean (±SEM) Corpus Luteum diameter in llamas induced to ovulate in the presence of a preovulatory follicle 7–10 or > 10 mm in diameter. Combined data irrespective of ovulation induction treatment (OIF/NGF or GnRH). Values with no common superscripts (a,b) are different (P < 0.05).

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a POF between 7 and 10 mm or >10 mm were grouped according to treatment (Fig. 2).

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>10 mm

OIF/NGF (n = 32)

Based on the results of the present study OIF/NGF administration induced a significantly greater increase in plasma progesterone concentration than that of the GnRH regardless of the preovulatory follicle diameter at the time of treatment. Also, it was demonstrated that preovulatory follicle diameter affects CL diameter; however, this event did not impact on the CL function in llamas. The results of this study confirm those of previous camelids reports where a consistently similar ovulatory response has been obtained when comparing the effect of OIF/NGF with a standard ovulatory dose of GnRH in llamas (Adams et al., 2005; Silva et al., 2011a; Ulloa-Leal et al., 2014) and alpacas (Huanca et al., 2012; Kershaw-Young et al., 2012). These studies have used either non purified (i.e. raw seminal plasma; Adams et al., 2005; KershawYoung et al., 2012; Ratto et al., 2005, 2006b; Silva et al., 2011b) or purified OIF/NGF (i.e. seminal plasma protein fractions or highly purified protein; Ratto et al., 2010,

Fig. 2. Mean (±SEM) plasma progesterone concentration in llamas induced to ovulate with either 50 ␮g of gonadoreline acetate or 1 mg of purified llama OIF. Combined data irrespective preovulatory follicle diameter (7–10 mm or ≥10 mm). Values with no common superscripts (a,b) are different (P < 0.05).

2011a; Silva et al., 2011a, 2012), and more recently recombinant ␤-NGF (Kershaw-Young et al., 2012; Ratto et al., 2012). The first studies evaluating the presence of an ovulationinducing factor in llama seminal plasma used a relatively conservative dose of seminal plasma (1 ml) based on the reported average volume of the ejaculate in alpacas and llamas (2–3 ml; Lichtenwalner et al., 1996). The OIF/NGF dose used in the present study was defined based on several studies (Silva et al., 2011a, 2012; Ulloa-Leal et al., 2014) that have demonstrated that the administration of 1 mg of purified llama OIF/NGF consistently induces an ovulatory response above 90%. We have hypothesized that this factor may act as an endocrine molecule (Ratto et al., 2012) since it is rapidly absorbed from the uterus into the systemic circulation after intrauterine deposition (Ratto et al., 2005) and acts at the hypothalamic GnRH-releasing neurons (Silva et al., 2011a). Based on the above, the definition of a standard ovulatory dose of OIF/NGF, that mimics the actual amount absorbed from the llama uterus after copulation warrants


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further investigations; however, Tanco et al. (2011) already demonstrated that doses representative of the amount of OIF/NGF present in 1/25 (500 ␮g of purified OIF) to 1/100 (125 ␮g of purified OIF) of a llama ejaculate induce an ovulatory response ranging from 70 to 90%, with a significant decrease occurring after administration of a dose of 60 ␮g of purified protein. In the present study, preovulatory follicle diameter did not affect ovulatory rate. As demonstrated by early studies in South American Camelids (Bravo et al., 1991) the ovulatory response is dependent upon follicle size, being drastically reduced in the presence of follicles between 4 and 5 mm in diameter, but with preovulatory follicles ≥7 mm in diameter fully responsive to the ovulatory stimulus. Results of the present study support these previous observations, further demonstrating that growing follicles between 7 and 10 or >10 mm in diameter have similar ovulatory potential in llamas. Although POF diameter did not influence ovulatory response, it did affect CL size, suggesting an association between POF and CL growth in llamas, similar to that described in ruminants. Several studies in cattle (Binelli et al., 2009; Busch et al., 2008; Perry et al., 2005; Vasconcelos et al., 2001), buffalos (Pandey et al., 2011) and sheep (Murdoch and Van Kirk, 1998) established a consistent relationship between POF diameter and CL size and function. For a normal CL development, preovulatory follicles must have adequate numbers of granulose cells and LH receptors on granulose and theca cell populations; additionally, granulose cells must be capable of synthesizing adequate amounts of progesterone after luteinization (McNatty et al., 1979). Interestingly, in the present study POF diameter did not influence plasma progesterone concentration after induction of ovulation. Similar observations have been made in cows that ovulated spontaneously, suggesting that under this condition other factors besides POF size influence CL function (Perry et al., 2005). These authors suggest that the intrafollicular concentration of estradiol could play a role in the preparation of follicular cells for luteal formation and function, through direct effects on granulose cell proliferation (Parrot and Skinner, 1998), acquisition of LH receptors (Wang and Greenwald, 1993) and aromatase activity (Reilly et al., 1996). Llamas in the present study, with the presence of a preovulatory follicle 7–10 or ≥10 mm in diameter, may have attained necessary intrafollicular estradiol concentrations to prepare follicular cells for luteinization regardless of follicular size. The former is supported by the strong correlation between follicle diameter and intrafollicular concentration of estradiol demonstrated in camels (El-Shahat et al., 2013), with medium (4–9 mm) and large (10–20 mm) follicles having greater concentration of this steroid hormone compared to small (1–3 mm) follicles. Whether or not a similar physiological mechanism applied for llamas or alpacas is a subject to be addressed in further investigations. Additionally, several authors have established an association between POF size and subsequent fertility in ruminants (Lamb et al., 2001; Mussard et al., 2007; Perry et al., 2005; Pandey et al., 2011; Vasconcelos et al., 2001), suggesting that the effect may be due to: (a) a higher

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increase in progesterone production by de CLs originating from larger follicles, (b) improved oocyte competence in those larger follicles, or (c) the association of both. The relationship between POF diameter and pregnancy rate in camelids has not been studied, but an increased competence is also reported for oocytes obtained from larger follicles in llamas (Trasorras et al., 2009) and camels (Kathir et al., 2007). As little is known about the influence of preovulatory follicle size on pregnancy rate after natural (i.e. mating) or pharmacological (i.e. GnRH) induction of ovulation in llamas, this aspect also warrants further studies. Under similar follicle size ovulation induction with either 50 ␮g of gonadoreline acetate or 1 mg of purified OIF/NGF did not affect CL diameter at Day 8 after treatment. Similar CL size has been also described after comparisons made between natural mating and GnRH administration (Ratto et al., 2006a). However, it has also been reported that 1 ml of homologous seminal plasma tended to induce (Adams et al., 2005) or induced (Huanca et al., 2012) CLs with greater diameter in llamas and alpacas respectively. The differences reported among studies may be related to variations in the total amount of OIF/NGF administered. In this regard, Tanco et al. (2011) demonstrated a doseresponse effect of OIF/NGF on CL diameter after induction of ovulation. Regardless of POF diameter at the time of treatment, llamas treated with 1 mg of purified OIF/NGF had greater plasma progesterone concentration at Day 8 compared to those treated with GnRH, confirming preliminary reports of luteotrophic effects of this molecule (Adams et al., 2005; Q4 Silva et al., 2011a; Tanco et al., 2011; Ulloa-Leal et al., 2014). The preovulatory LH surge initiates a series of molecular changes on the granulose cell population inducing the early stages of luteinization and CL formation. The prolonged LH surge consistently described after the administration of whole seminal plasma (Adams et al., 2005) or purified OIF (Ratto et al., 2011a; Silva et al., 2011a; Tanco et al., 2011) compared to that observed in GnRH-treated llamas, has been associated with higher plasma progesterone concentration in those females (Adams et al., 2005; Silva et al., 2011a). Recent studies using Color/Power Doppler Ultrasonography suggest that the luteotrophic effect of OIF/NGF may be related to the increase in preovulatory follicle vascular irrigation and/or to a similar effect observed during early CL development (days 4 and 6 after i.m. treatment administration (Ulloa-Leal et al., 2014)). These authors suggest that the increase in the vascular area of the follicle wall may be mediated by the treatment-induced LH surge. In mammals, the preovulatory and early CL development stages are characterized by an active angiogenic process (Reynolds et al., 2000). In this regard, the preovulatory LH surge is the main signal for up-regulating vascular endothelial growth factor (VEGF) that induces angiogenesis by stimulating the proliferation of endothelial cells of preexisting capillaries (Dvorak, 2002). Interestingly, several studies have demonstrated that NGF affects blood vessel area directly (Julio-Pieper et al., 2006) or indirectly by induction of expression of angiogenic factors such as VEGF and TGF␤1 at follicular level (Julio-Pieper et al., 2006, 2009). The above evidence suggests a biological mechanism


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by which OIF/NGF of seminal origin may affect CL function in llamas. Since plasma progesterone concentration during early luteal phase is essential for the establishment of pregnancy in several species including camelids, the administration of OIF/NGF as an ovulation-inducing agent instead of GnRH could become a pharmacological alternative strategy to improve llama fertility. Studies comparing the administration of 1 ml of alpaca seminal plasma vs GnRH administration, as ovulation-inducing agents, as part of artificial insemination (AI) and embryo transfer (ET) programs in alpacas (Huanca et al., 2012) could support this notion. We conclude that OIF/NGF treatment stimulates a greater increase in plasma progesterone concentration compared to that observed in llamas treated with GnRH, regardless of preovulatory follicle size at the time of treatment; and also that POF diameter affects CL size but not function in this species.

Conflict of interest None.

Acknowledgements

Research supported by the Chilean Research Council, Fondecyt Regular 1120518 and Proyecto Interno DGIP-UC 453 Q6 Temuco PF-2012-3-10 awarded to M Silva. 454 Q5 452

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Effect of aspiration of the preovulatory follicle on luteinization, corpus luteum function, and peripheral plasma gonadotropin concentrations in the mare.

Ovulation of the preovulatory follicle originating from the first-wave dominant follicle leads to formation of an active corpus luteum.

Corpus luteum function in ageing inbred mice.

Experimental aspects of corpus luteum function.

[Pregnancy onset without corpus luteum function?].

Corpus luteum function following subtotal hysterectomy in the prepuberal gilt.

[The corpus luteum reactivation test (CLR-test): a simple method for the characterization of the corpus luteum function (proceedings)].

nerve growth factor present in the seminal plasma of llamas.

Role of corpus luteum function in embryo implantation.

In vitro developmental competence of bovine oocytes: Effect of corpus luteum and follicle size.

[Proceedings: Influence of exogenous on corpus luteum function].

Effect of post-ovulatory administered oestrogens on corpus luteum function.

On the glandular origin of seminal plasma lipids in man.

Conversion of intraovarian patterns from preovulation to postovulation based on location of dominant follicle and corpus luteum in heifers.

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum.

Characterization and isolation of a high-density-lipoprotein-binding protein from bovine corpus luteum plasma membrane.

Ruptured corpus luteum with hemoperitoneum.

Early development and function of the corpus luteum and relationship to pregnancy in the buffalo.

The effect of intra-uterine prostaglandin F-2alpha on corpus luteum function in the human.

Variable tolerance of the developing follicle and corpus luteum to gonadotropin-releasing hormone antagonist-induced gonadotropin withdrawal in the human.

Angiogenesis in The Ovary - The Most Important Regulatory Event for Follicle and Corpus Luteum Development and Function in Cow - An Overview.

Recurrent hemorrhage from corpus luteum during anticoagulant therapy.

Massive intraperitoneal hemorrhage from ruptured corpus luteum during anticoagulant therapy.

Isolation of endothelial cells and pericytes from swine corpus luteum.